Protocal

Introduction There are many ways to measure the amount of a substance – if we have a relatively large amount, such as a spoonful of sugar, we can find its mass with a balance or find its volume by putting it in a measuring spoon. However, when we are trying to measure smaller quantities, or quantities that may be dissolved in a solution, we need to find another method for determining how much we have. For instance, it would be difficult to measure the amount of red food coloring we have in a bottle of Storage by weighing the bottle of Storage because it is mixed with so many different other molecules and water.

We could, if we had a lot of time, allow all the water to evaporate and chemically separate the red pigment from the other molecules so that we could weigh Just the red pigment on a balance. That would be a lot of work. However, there is a simpler way. Because some molecules absorb certain wavelengths of light, we can measure how much light they absorb at a certain wavelength. By comparing the amount of light absorbed by an unknown sample to the amount of light absorbed by a series of samples at known concentrations standards), we can determine how much is in our unknown sample.

Because the amount of light absorbed is directly proportional to the amount of our substance of interest (red coloring in Storage, for instance) we can calculate how much of the substance is in our solution if we know how much light is absorbed at a particular wavelength. Today, you will use a series of standards to construct a standard curve that will allow you to determine the concentration of an unknown sample. Next week, we will use a very similar procedure to determine the concentration of phosphate and nitrates in the river water. Your Job this week is to learn how to use the 1.

Turn your spectrophotometer on so that it can warm up. Make sure that the sample chamber is empty and the lid is closed. 2. Prepare your standards by making a serial dilution. A serial dilution is a way of making a set of solutions that decrease in concentration by a similar factor. Here, we will be diluting our stock solution by a factor of 2. Make the solutions in order going from tube 1 to tube 5. Tube number 3. Using the spectrophotometer: To take a reading, first press the Lambda key. This allows you to choose the wavelength that we will using to measure absorbency.

We will be using a wavelength of 664 NM for today because this is the wavelength that is most absorbed by our blue pigment. Enter the wavelength, and follow the directions on the screen. When the screen says that it is ready to measure, insert a cavetti that contains 2 ml of water (the little square tubes) with the arrow facing you. This is your blank, and will set the machine to zero. Press the”measure blank” button. The screen should tell you that the absorbency is O. Next, put 2 ml of the standard solution in tube 1 into the machine, and press “read sample button”. This should give you a umber larger than O but less than 1 .

Record the absorbency value for 2 ml of standard solution from each tube. Record your standard solution values in the chart below: Tube Absorbency value 4. Now, take the absorbency readings of your 3 unknown samples and record the absorbency values below. Unknown Unknown A Unknown B Unknown C 5. To be able to calculate the concentration of the blue pigment in each of the unknowns, you will need to make a graph of the absorbency value of the standard and the concentration of the standard. Abs Concentration of standard (MGM/ml) Now, plot your unknowns on the graph as well.

What is their concentration? Unknown I will show you as a class how to plot these values in Excel and determine the equation of the line. You can then use the equation of the line to calculate the concentration of any unknown for this pigment. Post laboratory assignment Working with your group, create a graph of your standard curve data in Excel. Be sure to include the following parts: 1 . A caption under the graph describing what the graph is about 2. Labels for the x and y axes 3. A line of best fit and the equation of the line Turn in one printed copy of the above for your entire group.